DMD – deletions in entire exons of the dystrophin gene – amplify exons using PCR and run through gel

Turner Syndrome= no Y chromosome

so detect with either chromosome painting, or PCR with Y-specific probes.

Patients, however, may have XY in the gonads, so you can detect gonad cells with Y-specific probe in PCR.

SSCP Analysis

unlike regular gel electrophoresis (in which double-stranded DNA travel through the gel), the DNA strands in a SSCP test are denatured, separated and run through a gel where they cannot reanneal. Only in this way can they form hairpin loops, and hairpin loops can affect their speed through a gel.

Different mutations form different hairpin loops (“conformations”) hence they are called single-strand conformational polymorphism

To do the SSCP test, compare how a Test DNA (with hairpin loops) vs. Control DNA (with hairpin loops) will run through the gel!

Advantages vs. Disadvantages

Advantages: You can test many exons at once.

Disadvantages: Not all point mutations will affect secondary structure.

DHPLC Analysis

like SSCP except you hybridize a single-stranded mutant strand with a normal strand, and they cause “kinks” that causes the whole thing to run differently in the gel.

CGH Analysis

It’s called Comparative Genomic Hybridization, because you are comparing the mutant with the control ‘s genome, how they compete with each other to hybridize to the array.

If much more Control DNA than the Test DNA hybridizes to the BACs on the array then it means that there is no Test DNA at this point of the chromosome, and therefore there is a deletion.

2. GENOMIC DISORDERS

During DNA Replication,

Backwards slippage → causes insertion

Forwards slippage → causes deletion

If slip not a multiple of 3, then frameshift mutation!

Imbalance in Protein Production causes problems! For every deletion there should also be a corresponding duplication! Hence Smith-Magenis vs. Potocki, or HNPP vs. Charcot-Marie-Tooth.

Maple Syrup Urine Disease –Defect in ketoacid decarboxylase. Excrete VIL (valine, isoleucine, and leucine) in urine. Common among Mennonites. Mnemonic: Think of Mennonites making maple syrup in their Village (VIL).

BF blocks XIST from inactivating Xa. This is why X chromosome in guys are not inactivated.

Gene inactivation doesn’t inactivate every gene. It doesn’t inactivate the PAR but it also doesn’t inactivate 15% of the rest of the X chromosome, and another 10% only sometimes inactivates.

Inactivation is always random, but sometimes it can look as if it is not, because sometimes cells with a mutation on the Active X may be selected out, leaving the good ones behind to populate the body.

X-linked Dominant disorders:

Craniofrontonasal Syndrome = loss of function in EFNB1, wide eyes. Females more affected than males, even though it’s X-linked!!

During gametogenesis, old imprints are erased, and re-established in gametes.

Here, the trisomic conceptus duplicates, but there is a nondisjunction that causes it to separate into 4n and 2n. The 2n keep multiplying to develop into a baby. Some will turn out normal. Others will be uniparental disomy.

Uniparental Isodisomy – nondisj
unction happen in meiosis II of gamete, followed by loss of chromosome after fertilization – both alleles from one parent, but identical.

Uniparental Heterodisomy – nondisjunction happen in meiosis I of gamete, followed by loss of chromosome after fertilization – both alleles from one parent, but different.

Angelman’s Syndrome vs. Prader-Willi Syndrome

Normally, UBE3A is maternally expressed, while father’s is imprinted. You get Angelman’s when mother’s allele is deleted or when there is paternal uniparental disomy.

Normally, SNRPN is paternally expressed, while mother’s is imprinted. You get Prader-Willi when father’s allele is deleted or when there is maternal uniparental disomy.

Detecting Angelman’s Syndrome vs. Prader-Willi on a gel

Prader-Willi patient has maternal band on a gel (because it has deletion in paternal chrom 15)

Angelman patient has paternal band on a gel (because it has deletion in maternal chrom 15)

Angelman’s patients sometimes have OCA (albinism) because on top of not expression UBE3A, father also has a mutated P gene.

Uniparental disomy of entire genome (not just a few genes) can lead to hydatidiform moles (ovarian teratoma)

Exons 1-13 of GNAS Gene encodes for production of alpha subunit of Gs protein, needed for PTH Hormone Response.

In Renal Proximal Tubule, you need just mother’s expression of 1-13 (because father’s is imprinted). If mutation is in mother, then you get PTH resistance

In all other tissues, you need both mother’s and father’s expression of 1-13. If mutation in either, then you get AHO.

Note in the diagram that PHP1A has both AHO and PTH resistance.

Note that PHPIB is due to imprinting defect, not mutation.

PHP vs PPHP

PHP1a = AHO w/ PTH resistance

PHP1b = PTH resistance w/o AHO

PPHP = AHO w/o PTH resistance

7. LINKAGE ANALYSIS

How to Identify a Mutation through Linkage Analysis

Collect sample DNA from all affected individuals and their families

Find markers over entire genome

Assess each marker to see how close it is to reflecting the behavior of the mutated gene (which we don’t know where it is). Do LOD score to figure this out. Find the markers with the least recombination frequency. Find the region of the marker and look for candidate mutated genes there.

Do SSCP analysis of each exon in each gene. Find which gene/exon has a band shift.

After narrowing in, do sequence analysis to find the exact mutation.

8. POPULATION GENETICS

Carrier frequency can “artificially” increase via founder effect.

Autosomal dominant disorders are selected. Recessive alleles stay under the radar because of the time they don’t affect the phenotype to be selected. X-linked recessive alleles only get selected when they are in males.

9. MULTIFACTORIAL DISEASES

SOD1 mutation causes ALS, but CNTF +/- can affect the age of onset (but only if there is already a SOD1 mutation to begin with)

Digenic Inheritance = retinitis pigmentosa caused by being heterozygous in two different genes. If you are only heterozygous in one of those genes, you won’t get the disease.

MTHFR = Methylene Tetra Hydro Folate Reductase → this gene encodes for an enzyme needed by the body to process folate. If a pregnant woman has a missense mutation in her MTHFR gene, it can cause her to be deficient in folate. If she ever gets pregnant, she can be at risk of having low folate, and therefore her fetus has a risk of neural tube defect. She needs to eat more than the recommended 400 micrograms per day of folate to decrease her baby’s risk of neural tube defect.

It’s the susceptibility to the disease that is the continuous character, not the disease itself (that is continuous, like height). You either have diabetes or you don’t, but your susceptibility is continuous.

Heritability

H=0 means disease is completely environmental.

H=1 means disease is completely genetic.

Most multifactorial diseases have Heritability of 0.4-0.7

10. NON-PARAMETRIC LINKAGE ANALYSIS

You can use these as association markers when doing Linkage Analysis:

SNPs – most stable, and best kind of marker to use – doesn’t keep changing from generation to generation like CA-repeats.